A common objective in biological and pharmaceutical research is the detection and identification of drug metabolites. Biological samples or fluids may be subjected to liquid chromatography/mass spectrometry (LC/MS) analysis for detecting the presence of, as well as for identifying specific, metabolites. The biological samples may include plasma, bile, spinal fluid, joint fluid, other bodily fluids, tissue extract, urine, and fecal extract for example.
Detection and identification of metabolites with low concentration in the biological samples, however, is often difficult due to significant interference from endogenous species (ions). A metabolite ion signal in a mass spectrum may be embedded among ion signals of endogenous ions. As a result, a species of interest may not be easily identifiable even by those skilled in LC/MS analysis. For example, a metabolite ion may be isobaric (i.e. having the same nominal, or integral, mass-to-charge ratio) with a naturally occurring biological substance. LC/MS data from a high resolution instrument can assist in distinguishing the metabolite from an endogenous isobaric interference.
In pharmaceutical development, the detection and identification of drug impurities and degradants is also a common task. Pharmaceuticals (i.e. drug products) may be subjected to LC/MS analysis for detecting the presence of, as well as for identifying specific impurities or degradants. The bioavailability of relatively insoluble drugs can be greatly enhanced by the addition of polymeric emulsifying agents such as Cremophor and polyethylene glycol (PEG) to the dosage form.
However, detection and identification of metabolites, impurities and degradants arising from drug products formulated in complex excipients, for example polymeric emulsifying agents, is difficult due to significant interference from such agents. Due to their complex or polymeric nature, such additives or excipients produce complex patterns in the mass spectra of the samples containing the agents. A drug-related impurity ion signal that is the target of analysis in a mass spectrum may be embedded among ion signals of polymeric ions, and thus cannot accurately be detected. It is therefore desirable to exclude such excipient signals during the detection and identification of the desired targets.
Detection and identification of drug metabolites or impurities and degradants for determining the effectiveness or quality of drugs is highly desirable. Existing techniques such as background subtraction are inadequate for the desired detection and identification of either drug metabolites in biological samples, or impurities and degradants that may occur in the sample in combination with polymeric agents in the drug formulation. An improvement over existing techniques is therefore necessary to more selectively identify metabolites and drug impurities and degradants in biological samples or in complex formulations, especially those occurring at low concentrations or with unknown molecular identities.
Accordingly, the present invention provides improved methods for detecting and identifying metabolites in biological samples and impurities in drug samples formulated with emulsifying agents.
A mass defect filter may be utilized for obtaining the desired selectivity in detection and identification of drug metabolites and impurities/degradants.